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© Copyright 2004 by the American Chemical Society

Volume 47, Number 1

January 1, 2004

Perspective The Discovery of Ezetimibe: A View from Outside the Receptor John W. Clader† Cardiovascular and CNS Medicinal Chemistry Department, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033-0539 Received June 9, 2003

Introduction The term “rationale drug design” has been used for more than 30 years to describe a marriage of experiment and technology intended to promote a more directed approach toward drug discovery, often focusing on whatever the hottest technology of the time might be.1 While the technology has changed over the years, the underlying goal has always been to provide a level of insight that would permit researchers to focus on compounds tailored to specific molecular targets with known function, in essence, to reduce the element of chance in the drug discovery process. The discovery of captopril is often cited as an early example of rational drug design, where a detailed knowledge of both the pharmacological rationale as well as the structure and function of the molecular target allowed a targeted synthesis of compounds specifically designed to elicit the desired effect.2 The decades that followed this discovery have seen a procession of technological initiatives designed to provide an ever more detailed view of structure and function, of how potential drug molecules might interact with their molecular targets, and of how the effects they elicit might impact disease. The promise of these developments was eloquently captured in Strader’s 1996 editorial in this journal,3 “The View from Inside the Receptor”. Increasingly, scientists would be able to view the interaction of small molecules from the perspective of the receptor, with a detailed understanding of what residues were key to providing affinity and function and how these residues interacted with a particular ligand. The pace of developments has not † Phone: 908-740-3478. Fax: 908-740-4563. E-mail: john.clader@ spcorp.com.

slowed in the ensuing years and in many ways has progressed even beyond that vision. Today, advances such as genomics and proteomics promise to tell us not only how drugs interact with their targets but also which targets are most relevant to human disease. The discovery of the novel cholesterol-lowering agent ezetimibe represents a distinct outlier from these trends.4 As the first new therapy for treatment of hypercholesterolemia since the discovery of the statins, ezetimibe represents a significant discovery by any measure, but the manner in which it was discovered was also clearly orthogonal to the technological currents of the time. Not only was it discovered without a clear understanding of the molecular target, even the existence of the target itself was unknown at the time the effort began. Even as the discovery process continued, the nature of the target could only be inferred from the existence of a consistent pattern of structure-activity relationships. There are several excellent review articles on the biology of ezetimibe and the results from clinical trials.5 This perspective will focus on the unusual process that led to its discovery and what lesson can be gleaned from this process. Discovery of 1,4-Bis(4-methoxyphenyl)-3(3-phenylpropyl)azetidin-2-one 14 (SCH 48461): The Prototype Azetidinone Cholesterol Absorption Inhibitor The discovery program that ultimately led to ezetimibe began as a program to discover novel ACAT (acylcoenzyme A cholesterol acyltransferase) inhibitors.6 Although ACAT was known to be involved in a variety of cholesterol trafficking events, including cholesterol absorption in rodents, the relevance of ACAT in non-

10.1021/jm030283g CCC: $27.50 © 2004 American Chemical Society Published on Web 12/09/2003

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Figure 1. Prototype ACAT inhibitors.

Scheme 1. Design and Synthesis of Early Azetidinones

rodent species was still unclear at the time this program began. Nonetheless, a variety of structural classes were known to be potent ACAT inhibitors in vitro and to be active in rodent animal models that reflect a potential for lowering cholesterol levels. Among these models was the cholesterol-fed hamster.7 A high-cholesterol diet dramatically increases liver cholesteryl ester (CE) levels in these animals, making them especially sensitive to ACAT inhibition. By contrast, serum cholesterol (SC) levels are not dramatically changed by cholesterol feeding in these animals, and most ACAT inhibitors have minimal effect on serum cholesterol levels. Figure 1 shows the in vitro and in vivo profiles of some typical early compounds from this effort, 1 and 2,8 as well as a reference ACAT inhibitor 3 (CI-976).9 Consistent with a well-defined molecular target, ACAT inhibitors displayed clearly defined structure-activity relationships in these models that logically followed from structural changes. Thus, proper conformational constraint of 1 led to indane 2, which showed both a significant increase in in vitro potency and a commensurate improvement in potency in the cholesterol-fed hamster.8 In addition to 2, a number of alternative conformationally constrained analogues were prepared to probe

their impact on in vitro and in vivo potency. Among these was azetidinone 4 proposed by Burnett et al.10 These compounds were prepared by ester-enolate condensation to give azetidinone 5, which was then deprotected by CAN oxidation, reduced, and acylated with a variety of acids (Scheme 1). In practice, the ester-enolate condensation gave only a modest yield of the desired azetidinone 5 accompanied by a small amount of a byproduct 6, apparently derived from deprotonation of 5 followed by Claisen condensation with the ethyl phenylacetate starting material. Both the desired product 4 and the intermediate 5 and byproduct 6 were evaluated for in vitro activity against ACAT as well as for in vivo activity in the cholesterolfed hamster (Table 1).10 None of the compounds was a potent ACAT inhibitor, although compound 6 did show some modest ACAT activity with an IC50 of about 7 µM. Despite this relatively weak activity, 6 showed moderate in vivo activity in the cholesterol-fed hamster assay. This included effects on both CE and a modest but reproducible effect on serum cholesterol. Initial follow-up of this lead structure by Burnett and co-workers (Table 2) demonstrated that 6 was not an isolated finding, since

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Table 1. In Vitro and In Vivo Activity of the First Azetidinones

several analogues of 6 displayed a similar profile of weak ACAT activity accompanied by a modest but reproducible effect on CE and SC in the hamster.11 Even at this early stage, elements of a structure-activity relationship were apparent, such as the loss in activity with aliphatic derivative 9. Improving the ACAT activity of these compounds, such as with 11 which adds the 2,4,6-trimethoxy moiety of 3, had little or no impact on the in vivo activity of compounds. On the basis of this observation, Burnett et al. opted to disregard the ACAT activity of subsequent analogues and focus on optimizing the in vivo activity of compounds, guided solely by activity in the cholesterol-fed hamster. The absence of an in vitro assay clearly made this an extraordinarily challenging medicinal chemistry effort. Nonetheless, this effort culminated in the discovery of azetidinone 13 and its resolved form 14, the prototype azetidinone cholesterol absorption inhibitors and the starting point for all subsequent work. It is noteworthy that the difference between 13 and less interesting analogues such as 12 is the addition of a single well-placed methoxy group at the C4 phenyl. Figure 2 shows the in vitro and in vivo profile of 14.12 In addition to blocking accumulation of hepatic cholesteryl esters in the hamster, 14 reduces serum cholesterol levels in the cholesterol-fed rat, dog, and monkey. Of particular significance is the activity in the cholesterolfed rhesus monkey. Figure 3 shows the effects of 14 on rhesus monkeys fed a high-cholesterol diet for 4 weeks.12 Control animals show a profound hypercholesterolemia after 3 weeks that is completely blocked by 1 mg/kg dose of 14 admininistered in the diet. When the control animals are then treated with 1 mg/kg of 14, their cholesterol levels return to nearly baseline within 1 week, while withdrawal of drug causes a gradual rise in cholesterol levels over the same time period. While ACAT inhibitors are known to inhibit cholesterol absorption in rodents, such potent antihypercholesterolemic activity in nonrodents was unprecedented with ACAT inhibitors. Furthermore, Salisbury et al. demonstrated that ACAT inhibitors and 14 have different effects on intracellular cholesteryl levels.12 In these experiments, the ACAT inhibitor 3 blocked the accumulation of 14C-cholesteryl esters but had no effect on free cholesterol in the intestinal wall of cholesterolfed hamsters. By contrast, 14 inhibited the accumulation of both esterified and unesterified cholesterol. The

reduction in cholesteryl ester levels was due entirely to reduction in free cholesterol substrate, while ACAT activity remained essentially unchanged. Thus, both the in vitro data and the in vivo pharmacology suggested that the azetidinone cholesterol absorption inhibitors act via a unique mechanism that is upstream from ACAT. From Serendipity to Design: The Discovery of Ezetimibe A substantial part of the chemistry program that followed the discovery of 14 was devoted to understanding the nature of the molecular target. A complete discussion of the ensuing SAR is the subject of other reviews and is beyond the scope of this perspective.13 Although the evidence is clearly circumstantial, these studies showed that the effects of 14 and related compounds were consistent with interaction with a welldefined molecular target. Among the most compelling evidence was once again the effects of conformationally constrained analogues such as 15-18 (Table 3).14,15 While these data provided encouragement, the absence of an in vitro assay made it difficult to separate potential effects on intrinsic potency from effects on pharmacokinetics. The latter was confounded by the fact that 14 is extensively metabolized in vivo, making it unclear exactly what the active species is.16 To address this latter issue, Van Heek and colleagues devised a unique experimental protocol designed to determine if there were active metabolites and how they contributed to the overall in vivo profile of 14.17 The experiment was divided into two parts, both of which used an intestinally cannulated, bile-duct-diverted rat model (Figure 4). In the first part of the experiment, animals were dosed via intraduodenal cannula with 3H-14. Bile from these animals was collected via the bile-duct cannula to give the so-called “metabolite” bile, which on the basis of previous experiments was known to contain the majority of the metabolites of 14. Concurrently, control animals were dosed with vehicle, bile was collected, and then 3H-14 was added to this so-called “parent” bile to match the specific activity of the metabolite containing bile. In the second part of the experiment, both the metabolite bile and the parent bile were again dosed intraduodenally into a second group of diverted animals

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Table 2. Early Azetidinone Structure-Activity Relationships

along with 14C-cholesterol, and the counts of both 14C and 3H in various tissues were measured. In this way, both the pharmacological effects as well as the disposition of drug could be measured in a single experiment by following counts of 14C and 3H, respectively. Furthermore, because of the bile duct diversion, the pharmacological effects reflected primarily the activity of the species being dosed and not the effects of any subsequently produced metabolites. In a third control arm of part 2, parent bile was dosed into intact, undiverted animals, where formation of active metabolites could

still contribute to activity. Figure 5 shows the results of these experiments. While the parent bile reduced the appearance of 14Ccholesterol in both the plasma and liver, the metabolite bile was clearly more effective and reduced 14C levels comparably to the parent compound 14 in intact animals. These data strongly suggested that formation of one or more active metabolites plays a significant role in the in vivo activity of 14. Consistent with this, when 3H-14 bile was dosed, the majority of the tritium counts were recovered in the bile. However, when 3H metabo-

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Figure 4. protocol.

Intestinally cannulated bile-duct-diverted rat

Figure 2. Profile of 14 in cholesterol-fed animal models.

Figure 5. Distribution of 14C-cholesterol and 3H-14 metabolites in bile-duct-diverted rats.17

Figure 3. Effect of 14 in the cholesterol-fed rhesus monkey. Table 3. Conformationally Constrained Analogues of 14

lite bile was dosed, the majority of the counts remained in the intestinal wall and lumen. These data suggest not only that are there active metabolites but that these metabolites localize at the putative site of action more efficiently than 14 itself.

These data begged the question of the identity of the active metabolites. To address this, metabolite bile was fractionated by HPLC and each fraction was evaluated according to the paradigm described in part 2 above. The results of this experiment are shown in Figure 6. On the basis of the appearance of 14C-cholesterol in plasma, these data showed that the bulk of the activity resided in fraction 6, and subsequent analysis showed that this fraction was composed primarily of the glucuronide of compound 19, a phenolic metabolite of 14. To complete the experiment, the activities of the metabolite bile, crude fraction 6, and authentic 19 were compared in bile-duct-diverted rats (Figure 7). These data show that the activity of 19 is identical to that of fraction 6 and both are substantially more active than the crude mixture of metabolites. In total, these data strongly suggested that much of the in vivo activity of 14 was due to the formation of 19. Furthermore, they suggested that metabolism of 14 to 19 helped to localize the compound in the intestines at the putative site of action of the compound. The experiments by Van Heek et al. provided compelling evidence for the presence of at least one active metabolite of 14. Nonetheless, there were also other less prominent metabolites whose formation could either contribute to the activity of 14 or diminish it. To understand this, Rosenblum et al. prepared authentic samples of a number of known or putative metabolites that were evaluated for activity in the cholesterol-fed hamster (Table 4).

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design of a second-generation compound, namely, (1) premetabolize profitable sites of metabolism on the C4 aryl and the phenylpropyl side chain to improve activity, minimize plasma levels, and localize the compound in the intestines and (2) block unprofitable sites of metabolism to maximize activity and limit further oxidative metabolism. This strategy was in fact applied to a number of chemical series related to 1419 but most successfully by Rosenblum et al. to give azetidinone 26, now known as ezetimibe.20 Figure 8 compares the activity of ezetimibe to 14 in a number of cholesterol-fed animal models.21 In every case, but most dramatically in the monkey, ezetimibe is substantially more active than 14 and shows substantially lower plasma levels.

Figure 6. Efficacy of total bile extract (ext) and fractionated bile.

Figure 7. Efficacy of fraction 6 versus 19 in bile-duct-diverted rats.

Among the various putative metabolites of 14 were a variety of phenols produced by dealkylation of either or both of the methoxy groups or via aromatic hydroxylation.18 Both phenol 20 and bisphenol 21 showed substantial activity in the cholesterol-fed hamster, although neither was more active than 14 or 19, suggesting that metabolism on the N-aryl moiety was not required for activity. On the other hand, phenol 22 was less active than 14, suggesting that this route of metabolism might be detrimental to activity. In addition to the phenols, another route of metabolism involved hydroxylation of the 3-phenylpropyl side chain to produce alcohols and ketones. (S)-Alcohol 23 was substantially more active than 14, the (R)-isomer 24 was less active, and the corresponding ketone 25 had intermediate activity. This suggested that metabolism to the (S)-alcohol, if not required for activity, might improve the activity of the compound. Other combinations of these routes of metabolism were also investigated with similar results. On the basis of the combined observations of experiments in bile-duct-diverted rats and the activity of various putative metabolites, a strategy emerged for the

Synthesis A number of syntheses of ezetimibe and related compounds have been reported, several of which were utilized in the course of these investigations.22 Many of these are based on an Evans-type oxazolidinone condensation to establish the correct stereochemistry on the azetidinone ring. Scheme 2 shows a representative synthesis of ezetimibe. In addition to the use of the oxazolidinone, this synthesis also features a Corey oxazaborolidine reduction to set the (S)-stereochemistry of the side chain hydroxyl group. Ezetimibe and Statins While the activity of ezetimibe in cholesterol-fed animals was clearly impressive, it is noteworthy that these models involve diets that are substantially higher in fat and cholesterol than the animals’ normal chow diet. Despite the substantial activity of ezetimibe and other azetidinone cholesterol absorption inhibitors in these models, none of the compounds tested significantly reduced plasma cholesterol levels in animals fed a normal chow diet. Rather than being a liability, this observation in fact led to one of the most important aspects of the profile of ezetimibe. In considering the possible reasons for the lack of substantial effect on serum cholesterol in the absence of a high cholesterol diet, Davis reasoned that a cholesterol absorption inhibitor might stimulate hepatic HMG-CoA reductase activity. This could compensate for a reduced cholesterol load due to inhibition of intestinal cholesterol absorption. If this were the case, then coadministration of a cholesterol absorption inhibitor and an HMG-CoA reductase inhibitor should produce an enhanced reduction in serum cholesterol at doses that were less effective or ineffective as monotherapy. To test this hypothesis, Davis et al. administered ezetimibe (0.007 mg/kg) or lovastatin (5 mg/kg) to chow fed dogs over 14 days.23 While neither compound had a substantial effect on serum cholesterol alone, the combination produced a profound reduction in serum cholesterol (Figure 9). Similar experiments demonstrated a comparable effect in other species and with other statins. These data suggested that ezetimibe would be effective at reducing cholesterol levels in humans and would be particularly effective in combination with HMG-CoA reductase inhibitors. Clinical Results Human clinical trials with ezetimibe supported the expectations of animal studies with ezetimibe both as

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Table 4. Activity of Possible Metabolites of 14 in Cholesterol-Fed Hamster

monotherapy and in combination with statins.24 Table 5 shows the results of phase III human trials with ezetimibe as monotherapy. Ezetimibe produced a significant reduction in total cholesterol, LDL cholesterol, and triglycerides as well as a small but significant increase in HDL cholesterol. Figure 10 compares the effect of simvastatin or atorvastatin either alone or when coadministered with ezetimibe on LDL cholesterol. In each case, ezetimibe produced an additional 15-18% reduction in LDL cholesterol above that achieved by the statin alone.

Finally, Figure 11 compares the effect of a high dose of statin alone with a low dose of statin coadministered with ezetimibe. In each case, coadministration of ezetimibe and low-dose statin produced an equivalent reduction in LDL cholesterol as the high dose of statin alone. Combined, these data demonstrate that ezetimibe alone or coadministration with statins provides favorable effects on the major lipid parameters in patients with hypercholesterolemia. Conclusions Case histories of drug discoveries that have made extensive use of a novel technology are often used as evidence for the validity of that technology as a drug discovery tool. Remarkably, essentially none of the technologies often associated with “modern” drug discovery played a significant role in the discovery of

Figure 8. Comparison of structure and in vivo profile of 14 and ezetimibe.

Figure 9. Effect of ezetimibe and lovastatin in chow-fed dogs.

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Scheme 2. Representative Synthesis of Ezetimibe

Table 5. Ezetimibe Phase III Monotherapy Efficacy Results mean % change from baseline at endpoint treatment placebo (n ) 226) ezetimibe, 10 mg (n ) 666) a

LDL total HDL cholesterol cholesterol cholesterol triglycerides +0.4

+0.8

-1.6

+5.7

-16.9a

-12.5a

+1.3a

-5.7

Significantly different from placebo results (p < 0.01).

Figure 10. Ezetimibe coadministered with simvastatin or atorvastatin.

frame not out of line with more biochemically driven programs of that time. The most important lesson from this discovery may be that while the technological advances of the past decade have unquestionably had a profound and positive impact on the discovery process, they have not reduced the importance of the discovery scientist in shaping and guiding this process. Even as it becomes more focused and technologically driven, drug discovery is still an experimental science. It remains a risky but distinctly human endeavor where serendipity and the opportunity to capitalize on it still play an important role. Acknowledgment. Ezetimibe was discovered by a team of Schering-Plough scientists from many scientific backgrounds who shared a common dedication toward scientific excellence and close collaboration. Without all of them and the unfailing support of Schering-Plough management, this project could not have succeeded. The author is privileged to present this case history on their behalf. Special thanks are given to Drs. M. Van Heek and H. Davis for many helpful comments and suggestions. Biography

Figure 11. Effect of high- and low-dose statin coadministered with ezetimibe.

ezetimibe, although they may be important for our understanding of the molecular target and the implications of this finding on cholesterol management and cholesterol homeostasis. The absence of these tools clearly created a number of challenges that required creative solutions, but in the end the entire process from initiation of the discovery program to discovery of the first clinical candidate 14 and finally to the discovery of ezetimibe was still completed in about 6 years, a time

John W. Clader received his Ph.D. in organic chemistry in 1980 from Indiana University. After 2 years at the University of Notre Dame, he began his career in medicinal chemistry at Hoffmann-La Roche in Nutley, New Jersey. He moved to the Schering-Plough Research Institute in 1985, where he is currently Distinguished Research Fellow in Medicinal Chemistry. In addition to ezetimibe, his research interests at Schering-Plough have included potential treatments for Alzheimer’s disease and HIV infection as well as the application of chemoinformatics and other computer methods to facilitate drug discovery.

References (1) For some recent discussions on rational drug design, see the following. (a) Parrill, A. L., Reddy, M. R., Eds. Rational Drug Design: Novel Methodology and Practical Applications; American Chemical Society: Washington, DC, 1999. (b) Weiner, D. B., Williams, W. V., Eds. Chemical and Structural Approaches to Rational Drug Design; CRC Press: Boca Raton, FL, 1995. (c) Weiner, D. B., Williams, W. V., Eds. Biological Approaches to Rational Drug Design; CRC Press: Boca Raton, FL, 1995. (d) Ganellin, C. R.; Roberts, S. M. The Role of Organic Chemistry in Drug Research, 2nd ed.; Academic Press: London, 2003. See also ref 3.

Perspective (2) Cushman, D. W.; Chung, H. S.; Sabo, E. F.; Ondetti, M. A. Design of Potent Competitive Inhibitors of Angiotensin Converting Enzyme. Carboxyalkanoyl and Mercaptanoyl Amino Acids. Biochemistry 1977, 16, 5484-5491. (3) Strader, C. D. The View from Inside the Receptor. J. Med. Chem. 1996, 39 (1), 1. (4) Earl, J.; Kirkpatrick, P. Fresh from the pipeline: Ezetimibe. Nat. Rev. Drug Discovery 2003, 2 (2), 97-98. For early reports on the discovery of ezetimibe, see also refs 19 and 22. (5) (a) Darkes, M. J. M.; Poole, R. M.; Goa, K. L. Ezetimibe. Am. J. Cardiovasc. Drugs 2003, 3 (1), 67-76. (b) Bruckert, E. New lipidmodifying therapies. Expert Opin. Invest. Drugs 2003, 12 (3), 325-335. (c) Gylling, H.; Miettinen, T. A. Combination therapy with statins. Curr. Opin. Invest. Drugs 2002, 3 (9), 1318-1323. (d) Gupta, E. K.; Ito, M. K. Ezetimibe: The First in a Novel Class of Selective Cholesterol-Absorption Inhibitors. Heart Dis. 2002, 4 (6), 399-409. (e) Bays, H. Ezetimibe. Expert Opin. Invest. Drugs 2002, 11 (11), 1587-1604. (f) Meng, C. Q. Ezetimibe Schering-Plough. Curr. Opin. Invest. Drugs 2002, 3 (3), 427432. (6) Sliskovic, D. R.; Picard, J. A.; Krause, B. R. ACAT inhibitors: The search for a novel and effective treatment of hypercholesterolemia and atherosclerosis. Prog. Med. Chem. 2002, 3, 121171. (7) Schnitzer-Polokoff, R.; Compton, D.; Boykow, G.; Davis, H.; Burrier, R. Effects of acyl-CoA: cholesterol O-acyltransferase inhibition on cholesterol absorption and plasma lipoprotein composition in hamsters. Comp. Biochem. Physiol. 1991, 99, 665-670. (8) (a) Clader, J. W.; Berger, J. G.; Burrier, R. E.; Davis, H. R.; Domalski, M.; Dugar, S.; Kogan, T. P.; Salisbury, B.; Vaccaro, W. Substituted (1,2-diarylethyl)amide acyl-CoA: cholesterol acyltransferase inhibitors: Effect of polar groups on in vitro and in vivo activity. J. Med. Chem. 1995, 38, 1600-1607. (b) Vaccaro, W.; Amore, C.; Berger, J.; Burrier, R.; Clader, J.; Davis, H.; Domalski, M.; Fevig, T.; Salisbury, B.; Sher, R. Inhibitors of Acyl CoA: Cholesterol Acyltransferase. J. Med. Chem. 1996, 39, 1704-1719. (9) Sliskovic, D. R.; White, A. D. Therapeutic Potential of ACAT Inhibitors as Lipid Lowering and Anti-atherosclerotic Agents. Trends Pharmacol. Sci. 1991, 323, 1289-1298. (10) Burnett, D. A.; Caplen, M. A.; Davis, H. R., Jr.; Burrier, R. E.; Clader, J. W. 2-Azetidinones as Inhibitors of Cholesterol Absorption. J. Med. Chem. 1994, 37, 1733-1736. (11) Clader, J. W.; Burnett, D. A.; Caplen, M. A.; Domalski, M. S.; Dugar, S.; Vaccaro, W.; Sher, R.; Browne, M. E.; Zhao, H.; Burrier, R. E.; Salisbury, B.; Davis, H. R., Jr. 2-Azetidinone Cholesterol Absorption Inhibitors: Structure-Activity Relationships on the Heterocyclic Nucleus. J. Med. Chem. 1996, 39, 3694-3693. (12) Salisbury, B. G.; Davis, H. R.; Burrier, R. E.; Burnett, D. A.; Boykow, G.; Caplen, M. A.; Clemmons, A. L.; Compton, D. S.; Hoos, L. M.; McGregor, D. G.; Schnitzer-Polokoff, R.; Smith, A. A.; Weig, B. C.; Zilli, D. L.; Clader, J. W.; Sybertz, E. J. Hypocholesterolemic activity of a novel inhibitor of cholesterol absorption, SCH 48461. Atherosclerosis 1995, 115, 45-63. (13) For two upcoming reviews on azetinidinone cholesterol absorption inhibitors, see the following. (a) Burnett, D. A. β-Lactam Cholesterol Absorption Inhibitors. Curr. Med. Chem., in press. (b) Clader, J. W. Ezetimibe and Other Azetidinone Cholesterol Absorption Inhibitors. Curr. Top. Med. Chem., in press. (14) Dugar, S.; Clader, J. W.; Chan, T.-M.; Davis, H. R., Jr. Substituted 2-Azaspiro[5.3]nonan-1-ones as Potent Cholesterol Absorption Inhibitors: Defining a Binding Conformation of SCH 48461. J. Med. Chem. 1995, 38, 4875-4877. (15) Dugar, S. Unpublished results. For a related series of unsaturated analogues, see the following. Rosenblum, S. R.; Huynh, T.; Afonso, A.; Davis, H. R., Jr. Synthesis of 3-arylpropenyl, 3-arylpropynyl, and 3-arylpropyl 2-azetidinones as cholesterol absorption inhibitors: Application of the palladium-catalyzed arylation of alkenes and alkynes. Tetrahedron 2000, 56, 5735-5742. (16) Yumibe, N. Unpublished results. See also refs 17 and 20. (17) Van Heek, M.; France, C. F.; Compton, D. S.; McLeod, R. L.; Yumibe, N. P.; Alton, K. B.; Sybertz, E. J.; Davis, H. R., Jr. In vivo metabolism-based discovery of a potent cholesterol absorption inhibitor, SCH 58235, in the rat and rhesus monkey through identification of the active metabolites of SCH 48461. J. Pharmacol. Exp. Ther. 1997, 283, 157-163. (18) Yumibe, N. Unpublished results. See also refs 17 and 20.

Journal of Medicinal Chemistry, 2004, Vol. 47, No. 1 9 (19) For representative examples, see the following. (a) McKittrick, B. A.; Ma, K.; Dugar, S.; Clader, J. W.; Davis, H. Jr., Czarniecki, M. Stereoselective Synthesis and Biological Activity of CIS Azetidinones as Cholesterol Absorption Inhibitors. Bioorg. Med. Chem. Lett. 1996, 6, 1947-1950. (b) Dugar, S.; Yumibe, N.; Clader, J. W.; Vizziano, M.; Huie, K.; Van Heek, M.; Compton, D. S.; Davis, H. R., Jr. Metabolism and Structure Activity Data Based Design: Discovery of (-) SCH 53079. An Analog of the Potent Cholesterol Absorption Inhibitor (-) SCH 48461. Bioorg. Med. Chem. Lett. 1996, 6, 1271-1274. (c) Kirkup, M. P.; Rizvi, R.; Shankar, B.; Shankar, B.; Dugar, S.; Clader, J. W.; McCombie, S. W.; Lin, S.-I.; Yumibe, N. (-)-SCH 57939: synthesis and pharmacological properties of a potent, metabolically stable cholesterol absorption inhibitor. Bioorg. Med. Chem. Lett. 1996, 6, 2069-2072. (d) McKittrick, B. A.; Ma, K.; Huie, K.; Yumibe, N.; Davis, H. R., Jr.; Clader, J. W.; Czarniecki, M.; McPhail, A. T. Synthesis of C3 Heteroatom-Substituted Azetidinones That Display Potent Cholesterol Absorption Inhibitory Activity. J. Med. Chem. 1998, 41, 752-759. (20) Rosenblum, S. B.; Huynh, T.; Afonso, A.; Davis, H. R., Jr.; Yumibe, N.; Clader, J. W.; Burnett, D. A. Discovery of 1-(4Fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)(4-hydroxyphenyl)-2-azetidinone (SCH 58235): A Designed, Potent, Orally Active Inhibitor of Cholesterol Absorption. J. Med. Chem. 1998, 41 (6), 973-980. (21) See refs 17 and 23. (22) (a) Homann, M. J.; Previte, E. Stereoselective microbial reduction for the preparation of 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3(4-fluorophenyl)propyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone. U.S. Patent 6,133,001, 2000. (b) Thiruvengadam, T. K.; Fu, X.; Tann, C.-H.; Mcallister, T. L.; Chiu, J. S.; Colon, C. Process for the synthesis of azetidinones and intermediates for use as hypocholesterolemics. Patent WO0034240, 2000. (c) Wu, G.; Wong, Y.; Chen, X.; Ding, Z. A Novel One-Step Diastereo- and Enantioselective Formation of trans-Azetidinones and Its Application to the Total Synthesis of Cholesterol Absorption Inhibitors. J. Org. Chem. 1999, 64, 3714-3718. (d) Wu, G.-Z.; Chen, X.; Wong, Y.-S.; Schumacher, D. P.; Steinman, M. A process for the enantioselective preparation of azetidinones using 3-hydroxy gammalactone. U.S. Patent 5,886,171, 1999. (e) Shankar, B. B. Process for preparing 1-(4-fluorophenyl)-3(R)-(3(S)-hydroxy-3-([phenyl or 4-fluorophenyl])-propyl)-4(S)-(4-hydroxyphenyl)-2-azetidinone. U.S. Patent 5,856,473, 1999. (f) Wu, G.-Z.; Chen, X.; Wong, Y.-S.; Schumacher, D. P.; Steinman, M. 3-Hydroxy gamma-lactone based enantioselective synthesis of azetidinones. Patent WO9745406, 1997. (g) Shankar, B. B.; Kirkup, M. P.; McCombie, S. W.; Clader, J. W.; Ganguly, A. K. Synthesis of an Optically Pure 3-Unsubstituted β-Lactam Using an Asymmetric Reformatsky Reaction and Its Conversion to Cholesterol Absorption Inhibitors. Tetrahedron Lett. 1996, 37, 4095-4098. (h) Burnett, D. A. Asymmetric Synthesis and Absolute Stereochemistry of Cholesterol Absorption Inhibitor, SCH 48461. Tetrahedon Lett. 1994, 35, 7339-7342. (23) Davis, H. R., Jr.; Pula, K. K.; Alton, K. B.; Burrier, R. E.; Watkins, R. W. The synergistic hypocholesterolemic activity of the potent cholesterol absorption inhibitor, ezetimibe, in combination with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in dogs. Metab., Clin. Exp. 2001, 50, 1234-1241. (24) (a) Dujovne, C. A.; Ettinger, M. P.; McNeer, J. F.; Lipka, L. J.; LeBeaut, A. P.; Suresh, R.; Yang, B. O.; Veltri, E. P. Efficacy and safety of a potent new selective cholesterol absorption inhibitor, ezetimibe, in patients with primary hypercholesterolemia. Am. J. Cardiol. 2002, 90 (10), 1092-1097. (b) Davidson, M. H.; McGarry, T.; Bettis, R.; Melani, L.; Lipka, L. J.; LeBeaut, A. P.; Suresh, R.; Sun, S.; Veltri, E. P. Ezetimibe coadministered with simvastatin in patients with primary hypercholesterolemia. J. Am. Coll. Cardiol. 2002, 40 (12), 2125-2134. See also ref 5. (c) Gagne, C.; Bays, H. E.; Weiss, S. R.; Mata, P.; Quinto, K.; Melino, M.; Cho, M.; Musliner, T. A.; Gumbiner, B. Efficacy and safety of ezetimibe added to ongoing statin therapy for treatment of patients with primary hypercholesterolemia. Am. J. Cardiol. 2002, 90 (10), 1084-1091. (d) Ballantyne, C. M.; Houri, J.; Notarbartolo, A.; Melani, L.; Lipka, L. J.; Suresh, R.; Sun, S.; LeBeaut, A. P.; Sager, P. T.; Veltri, E. P. Effect of Ezetimibe Coadministered with Atorvastatin in 628 Patients with Primary Hypercholesterolemia. Circulation 2003, 107 (19), 2409-2415.

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